The present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. More specifically, it relates to the use of a selected sugar as energy source in the culture media used during specific stages of somatic embryo development. The invention is especially suited for producing large numbers of clones of superior selections useful for reforestation.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are probably the most important commercial species of temperate North American timber trees. Similarly, Norway spruce (Picea abies (L.) Karst.) is probably the most important European softwood species. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted on cut-over or burned forest lands. For many years these seedling trees were grown using naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees discovered in the forests. These trees were selected for such heritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducing some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
A brief review of some of the most important work relating to the present invention will follow. This is intended to be representative only and is not fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full citations of the literature noted herein.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, potential genetic gain, and much lower probability of mutations. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage.
Earlier U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, and 5,236,841, herein incorporated by reference, describe improved methods of conifer embryogenesis. These also include extensive reviews of the most closely related literature. In the methods described in all of these patents, advanced early stage embryos (or "late stage proembryos"), defined as totipotent embryonic structures estimated to have least about 100 mostly undifferentiated cells, are transferred to and further cultured in a cotyledonary embryo development medium containing abscisic acid (ABA) as an essential growth hormone. It appears to be highly desirable during this stage to gradually reduce the level of exogenous ABA so that little or none is ultimately present. Other growth hormones; e.g., gibberellins, may also be used at this time. The ultimate product of this culturing step is somatic embryos resembling natural zygotic embryos in morphology.
It is well accepted that plant tissue culture is a highly unpredictable science. Sondahl et at., in published European Patent Application 293,598, speak directly to this point.
"Since each plant species appears to possess a unique optimal set of media requirements, the successful preparation and regeneration of a new species cannot be necessarily inferred from the successful regimens applied to unrelated plant species." PA1 "Despite progress, our knowledge of embryogenesis is still fragmentary. At present we cannot yet define the conditions necessary for embryogenesis . . . "
This statement can be carried even farther. Rangaswamy (1986) notes that the potential for embryogenesis is even genotype specific within any given species.
Compositions of the media used to initiate embryogenesis and induce embryo maturation are critical to success, regardless of the species being propagated. In particular, the type and level of the nitrogen source in the media and the presence or absence, composition, level, and timing of availability of growth hormones have been the key to success. It is also these very factors, particularly the hormones, that have proved to be so unpredictable. As one example, Ammirato (1977), conducted a study examining the effects of zeatin (a cytokinin), ABA, and gibberellic acid (GA.sub.3) on the yield and morphology of caraway (Carum carvi) somatic embryos. These hormones were present singly and in all possible combinations in the media used for the later stages of embryo development. He concluded that a change in level or presence/absence of any one of the hormones caused a ripple effect felt throughout the system due to unpredictable interactions between the various hormones. Lakshmi Sita (1985) summarizes her earlier work and that of others in promoting embryogenesis of sandalwood (Santalum sp.). Gibberellic Acid was found to be useful in inducing embryogenesis using shoot explants in either solid or liquid suspension cultures. Despite her success, which included successful production of converted plants, she again points to the lack of predictability of embryogenesis.
The same problem is again discussed by Evans (1984) who notes that growth hormones which affect the same process can either act independently or may interact in some fashion.
In general, as far as coniferous species are concerned, it appears that at least one exogenous auxin and usually a cytokinin are necessary hormones in a medium for the initiation of embryogenesis. While much work has been done studying the effect of the stimulatory growth hormones, and the effect of the nitrogen source in the media, little consideration has been given to the carbohydrate used as the carbon and energy source for the growing embryos. It has been known that various sugars were metabolized by developing embryos; e.g., U.S. Pat. No. 5,036,007 suggests that sucrose, glucose, fructose, maltose or galactose are metabolizable and suitable for osmotic potential control in cotyledonary embryo development media. These were used alone or in combination with poorly metabolized materials such as polyalkylene glycols. However, it has been generally assumed by most workers that sucrose or glucose were optimal.
Stuart et al., in U.S. Pat. No. 4,801,545, note that "maltose has been used in several studies of plant growth and differentiation without success" and cite several references to this effect. However, they found that about 90 mM of maltose in combination with at least one amino acid chosen from the group of proline, alanine, or glutamine increased the number of embryos produced in alfalfa cultures. In a closely related article, Strickland et al. (1987) noted that maltose, maltotriose, and soluble starch all acted to improve embryo morphology and conversion in alfalfa cultures. The presence of ammonium ion (NH.sub.4.sup.+) was said to be essential with maltose in order to see the improvement.
Uddin, in U.S. Pat. No. 5,187,092, describes somatic embryogenesis of loblolly pine using glucose or maltose in combination with abscisic acid in the mature embryo development medium. The data available in the Uddin patent are very limited. However, it appears that a two stage treatment in which the ABA level is increased and the auxin indolebutryic acid is added to the second stage is necessary if the claimed improvements are to be achieved. The high level of ABA and the stepwise increase are at odds with others in the field who have found that ABA is needed at relatively low levels which should preferably be decreased during the development period; e.g. as taught in U.S. Pat. Nos. 5,034,326 and 5,236,841.
Nagmani and Dinus ((1991) use a procedure similar to that of Uddin by employing maltose in the cotyledonary embryo development stage but with Douglas-fir rather than loblolly pine.
More recently, Beewar et at., in U.S. Pat. No. 5,413,930, teach the use of maltose in cotyledonary embryo development medium for Pinus species.
Tremblay and Tremblay (1991) explored various carbohydrate sources in the cotyledonary development (or maturation) medium for black spruce (Picea mariana) and red spruce (Picea rubens) embryos taken from a maintenance culture. Maltose was found to be about equivalent to glucose or sucrose for red spruce and much inferior to glucose or sucrose in promoting mature embryo development in black spruce. These investigators concluded that "different spruce species have varying carbohydrate requirements for the development of somatic embryos".
Schuller and Reuther (1993) looked at a similar selection of carbohydrate energy sources for developing embryos of European silver fir (Abies alba), although they substituted soluble starch for maltose. Soluble starch and lactose were found to be most effective at the later stages of cotyledonary embryo development.
Techniques to promote embryogenesis of numerous conifer genera are now well established. Research emphasis is now shifting to development of ways to scale up laboratory knowledge and techniques so that the process may become field operational on large scale. Yet many problems of a relatively fundamental nature still remain to be solved. One of these is improving somatic embryo quality and vigor. This is necessary so that germination to hardy plantlets and ultimate conversion to growing trees can be achieved at much higher percentages than has heretofore been possible. As workers gain more experience in conifer embryogenesis it has become evident that well formed advanced early stage embryos entering the development stage are a critical necessity for production of vigorous and well developed cotyledonary embryos. Reference might be made to U.S. Pat. No. 4,957,866 in this regard. The present invention is directed to this end.